Zn deficiency is recognized as a risk factor for pregnancy complications;however, the mechanisms underlying its teratogenicity are obscure. Aim 1 of this proposal is to characterize temporal events underlying Zn deficiency-induced alterations in growth factor metabolism and their contribution to cell proliferation, cell survival, and abnormal embryo development. We will determine if in 3T3 embryonic fibroblasts, cellular Zn deficiency results in increased PTEN phosphatase activity, decreased PIP3 levels, and decreased membrane recruitment/activity of Akt. We suggest that the diminution of RTK signaling through both the PI3K and MAPK pathways results in co-inhibition of Akt and ERK 1/2, respectively, which causes cell cycle arrest and conditions the cell for eventual apoptosis. Results will be compared to Pten-null cell lines, which should be less sensitive to Zn deficiency. Zn "rescue" involves inhibition of PTEN and the reactivation of both P13K and MAPK signaling pathways, which results in the down-regulation of the cell cycle inhibitor, p27/kip1, and the cytosolic sequestration and phosphorylation of the pro-apoptotic protein, BAD. We will determine if low cellular Zn results in a temporal decrease in RTK signaling downstream of ligand binding, giving rise to defective PI3K (PI3K-->Akt) and MAPK (Ras-->ERK) signaling in the somites and neural tube. Findings will be correlated with the spatio-temporal expression of the IGF-l-dependent antiapoptotic transcription factor TWIST. Using inhibitors and activators of the PI3K/MAPK pathways and Pten heterozygous mice, we will determine in embryos the contribution of each pathway to cell proliferation/survival under conditions of Zn deficiency or Zn rescue. We will determine to what extent the small embryo size and maldevelopment induced by Zn deficiency is mediated by IGF-1 deficiency (a function of decreased IGF-1 gene expression and altered IGFBP profiles) or defects in RTK signaling. The relative contributions of the IGF axis components to abnormal embryo development will be characterized in vitro by manipulating IGF and IGFBP levels and profiles using specific neutralizing antibodies and IGF analogues. In specific Aim 2, we will characterize the Zn deficiency-induced alterations in neural crest cell (NCC) migration and survival. We will determine if: a) Zn deficiency negatively affects NCC viability, proliferation and cell death in vivo and in vitro;b) NCC subjected to Zn deficiency in vivo and in vitro have impaired migration as a consequence of altered cytoskeletal organization and polymerization of microtubules;c) Zn deficiency alters cell: cell adhesion and cell: cell communication in NCC. The temporal expression of N-cadherin and the dynamics of focal adhesion formation will be determined using immunohistochemistry for cadherin and key focal adhesion components (talin, vinculin, FAK (focal adhesion kinase)). Gap junction integrity will be determined using Lucifer yellow microinjection;d) Zn deficiency alters the subcellular localization and temporal expression of the Rho-GTPases (Rho, Rac and Cdc42) as a result of reduced FAK signaling through PI3K, due to a Zn deficiency-induced increase in PTEN activity, leading to NCC detachment mediated cell death.